Human nipple areolar complex extracellular matrix scaffold and methods relating thereto

ABSTRACT

Methods of producing and methods of treatment using a nipple areolar complex extracellular matrix. A method for processing tissue comprises harvesting a human nipple areolar complex tissue, treating the tissue with a stabilizing medium, wherein the medium stabilizes the tissue during transport, decellularizing the tissue, and sterilizing the tissue to form a human nipple areolar complex extracellular matrix scaffold (ECMS). A method of treating an individual with a need for a nipple reconstruction comprises forming an ECMS and applying the ECMS to the individual for nipple areolar regeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/626,058, filed on Jun. 17, 2017 which is thenational phase filing of PCT/US2016/066024, filed on Dec. 9, 2016,entitled “Human Nipple Areolar Complex Extracellular Matrix Scaffold AndMethods Relating Thereto” which claims priority to U.S. PatentApplication Ser. No. 62/265,798, filed Dec. 10, 2015, entitled “NippleAreolar Complex Extracellular Matrix Scaffold”. The foregoing patentapplications are hereby incorporated by reference in their entirety forall purposes.

This application includes material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent disclosure, as it appears in thePatent and Trademark Office files or records, but otherwise reserves allcopyright rights whatsoever.

FIELD

The present invention is related to the creation of an extracellularmatrix scaffold from autologous or allograft tissue for use in humannipple areola tissue regeneration, reconstruction, or replacement.

BACKGROUND

Nipple areolar reconstruction is desired in individuals whom have lost anipple due to cancer, trauma, or congenital absence. Current options fornipple reconstruction include recreating the appearance of a nipple by aprosthetic silicone nipple that is adhered to the skin of the breastmound, surgical reconstruction with or without tattoo to match naturalnipple pigmentation, or a tattoo of a nipple.

Surgical reconstruction may be performed using a composite nipple graftfrom the contralateral nipple, a local skin flap, skin flaps withautologous graft augmentation, skin flaps with alloplastic augmentation,or skin flaps with allograft augmentation.

Composite nipple grafts use a portion of the contralateral nipple as atissue graft for the nipple reconstruction. This type of reconstructionrequires a contralateral nipple with adequate volume to accommodate adonation to the other side. There is also risk of donor site morbidity,nipple discoloration, and loss of sensation of the donor nipple.

Local skin flaps are most commonly used to recreate the appearance of anipple. Loss of projection occurs in all nipples created from surgicalflaps, due to retraction forces of the underlying tissue and contractionof scar tissue. To address loss of projection, surgical flaps may beaugmented with autologous tissue such as cartilage or fat. Autologoustissue donation requires a secondary surgical site with risk of donorsite morbidity. Surgical flaps may also be augmented with alloplasticsubstances such as silicone gel, hyaluronic acid, calciumhydroxylapatite (RADIESSE™), hydroxyapatite and tricalcium phosphate(CERATITE™), and polytetrafluoroethylene (PTFE). A major disadvantage ofalloplastic augmentation is risk of infection and or extrusion of thesubstance.

Xenograft tissue such as extracellular collagen matrix derived fromporcine small intestinal mucosa (Cook medical nipple reconstructioncylinder) may also be used to augment surgical flaps. However, this typeof material may not be used in individuals with sensitivity to porcinematerial or with irradiated skin. Acellular dermal allografts have beenused to augment surgical flaps. Allograft tissue has a higher rate ofincorporation with limited resorption, reducing risk of infection and orextrusion. Maintained structure and biochemistry of the tissue matrixallows for tissue integration into the graft, with native cellsrepopulating the matrix.

All surgical flap reconstructed nipples will flatten by 50-75% over 2years. In approximately 10-15% of reconstructions, the nipple will loseall projection. The most common reason for dissatisfaction followingnipple-areolar reconstruction is lack of projection, followed by colormatch, shape, size, texture and position. Only 13% of all patients weretotally satisfied with their reconstructed nipple areolar complex.Factors that may negatively affect projection include external pressureon the reconstruction, poor surgical design, thin skin and dermis, andlack of subcutaneous fat.

All current methods of nipple reconstruction only recreate theappearance of a nipple. The nipple is a site of specialized epidermissince it is glabrous, associated with a unique gland (mammary), hasdistinct patterns of epidermal stratification, and expresses uniquedifferentiation markers that are not present in trunk epidermis. Duringlactation, the nipple undergoes high mechanical strain as well asprolonged exposure to high moisture and digestive enzymes in saliva. Inhumans, the nipple has approximately 15-20 lactiferous ducts centrallylocated that are lined mainly by a two-layered stratified cuboidalepithelium. Tightly packed collagen bundles surround the ducts as wellas smooth muscle that is oriented parallel and circular to the ducts.The nipple has deep infolding of the epidermis into an extensivepapillary dermis with fine collagen bundles. The human areola hassimilar invaginations of the epidermis, although less dramatic. Thehuman areola has large sebaceous glands called Montgomery's tuberclesthat are thought to prevent sore or chapped nipples and secretepheromone substances that aid in nursing. The human nipple areolarcomplex is more pigmented than surrounding skin with melanocytesabundant not only in the epidermis, but also in the basal layer of thesebaceous gland and lactiferous ducts. It is thought that the growthfactor environment of the nipple areolar complex (NAC) tissue promotesmelanocyte survival in areas where they are typically not found.

Despite efforts to recreate cosmetic solutions for NAC tissuereplacement, there remains a significant need in the art for human NACreplacements capable of reproducing the complex physiological andmorphological characteristics of a human NAC.

SUMMARY OF THE DISCLOSURE

It is therefore an object of the present invention to provide a humannipple areolar complex extracellular matrix scaffold from autologous orallograft human nipple areolar tissue for use in human nipplereconstruction, regeneration, or replacement.

In one aspect of the present invention, a method for processing tissuecomprises harvesting a human nipple areolar complex tissue, treating thetissue with a stabilizing medium, wherein the medium stabilizes thetissue during transport, decellularizing the tissue, and sterilizing thetissue to form a human nipple areolar complex extracellular matrixscaffold (ECMS). In some embodiments, the harvesting step comprisesharvesting the tissue from a cadaver. In some embodiments, theharvesting step comprises harvesting the tissue from an individualduring a surgery.

In another aspect of the present invention, a method of treatmentcomprises identifying an individual with a need for a nipplereconstruction, forming a human nipple areolar complex extracellularmatrix scaffold (ECMS), and applying the ECMS to the individual. Theforming comprises harvesting a human nipple areolar complex tissue,treating the tissue with a stabilizing medium, wherein the mediumstabilizes the tissue during transport, decellularizing the tissue, andsterilizing the tissue to form an ECMS. In some embodiments, theindividual is an individual who has lost a nipple due to a cancer, atrauma, or a congenital absence. In some embodiments, the individual isan individual seeking a new nipple for a cosmetic reason. In someembodiments, the applying step comprises direct implantation of the ECMSonto the individual for in vivo cellular repopulation using theindividual's cellular responses. In some embodiments, the method furthercomprises, before the applying step, recellularizing the ECMS to yield arecellularized ECMS, wherein the applying step comprises applying therecellularized ECMS to the individual.

It is therefore an object of the present invention to provide a methodfor regenerating a nipple areolar complex comprising: harvesting anipple areolar complex tissue; treating the tissue with a stabilizingmedium, wherein the medium stabilizes the tissue during a transport; anddecellularizing the tissue; wherein a nipple areolar complexextracellular matrix scaffold (ECMS) is produced comprised of at leastdermal or subdermal layers. In another aspect a further step comprisessterilizing the nipple areolar complex ECMS. In another aspect theharvested nipple areolar complex is human and may be harvested tissuefrom a cadaver or from an individual during a surgery.

In another aspect, the decellularizing step comprises decellularizationusing one or more decellularization methods selected from the groupconsisting of: detergents, enzymes, salts, electrophoresis, andcombinations thereof. An additional step includes after application ofthe nipple areolar complex ECMS to an individual, the scaffold isrepopulated by cells in vivo. In another aspect the present inventioncomprises repopulating the nipple areolar complex ECMS with exogenouscells selected from the group consisting of: fibroblasts, epithelialcells, mammary endothelial cells, mammary epithelial cells, vascularsmooth muscle cells, bone marrow mesenchymal stem cells, adipose derivedstem cells, induced pluripotent stem cells, or combinations thereof.

In another aspect of the present invention, a method of treatmentcomprises: identifying an individual with a need for a nipplereconstruction; forming a nipple areolar complex extracellular matrixscaffold (ECMS), the forming comprising; harvesting a nipple areolarcomplex tissue; treating the tissue with a stabilizing medium, whereinthe medium stabilizes the tissue during transport; decellularizing thetissue to yield a sterile nipple areolar complex ECMS comprising atleast dermal or subdermal layers; and applying the nipple areolarcomplex ECMS to the individual.

The method of the present invention may be performed for an individualwho has lost a nipple due to a cancer, a trauma, congenital absence, ora cosmetic reason. In another aspect, the harvested nipple areolarcomplex tissue is human nipple areolar complex tissue.

In another aspect, direct implantation of the ECMS onto the individualis performed. The implanted decellularized ECMS may be repopulatednaturally by the patient's native cells, or may be populated byrecellularizing the ECMS to yield a recellularized ECMS, and wherein theapplying step comprises applying the recellularized ECMS to theindividual.

Such a recellularization step may further comprise recellularizing thenipple areolar complex ECMS with exogenous cells selected from the groupconsisting of: fibroblasts, epithelial cells, mammary endothelial cells,mammary epithelial cells, vascular smooth muscle cells, bone marrowmesenchymal stem cells, adipose derived stem cells, induced pluripotentstem cells, or combinations thereof.

It is another object of the present invention to provide a nippleareolar complex extracellular matrix scaffold (ECMS), comprising adecellularized nipple areolar complex ECMS, wherein the nipple areolarcomplex ECMS is capable of supporting regeneration of a nipple areolarcomplex when applied to a patient. The nipple areolar complex ECMS maybe derived from human nipple areolar tissue and may further be capableof supporting growth of melanocytes. The nipple areolar complex ECMS ofthe present invention comprises at least dermal layers.

In one aspect, the present invention has the potential for increasedsensation of the nipple area complex occurring within 2 years of ECMSincorporation and may further exhibit maintained projection of thenipple, retained structure after processing.

In one aspect of the present invention, the nipple areolar complex ECMSdoes not illicit a negative immune response when exposed to arecipient's cells. The nipple areolar complex ECMS may be applied to anindividual, and is capable of generating pigmentation of the nipple areacomplex.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in the detailed descriptionwhich follows, in reference to the noted plurality of drawings by way ofnon-limiting examples of exemplary embodiments, in which like referencenumerals represent similar parts throughout the several views of thedrawings, and wherein:

FIG. 1 depicts a flow chart of an embodiment of a method of fabricatinga human nipple areolar complex extracellular matrix scaffold from humannipple areolar complex tissue;

FIG. 2 depicts an embodiment of native human nipple areolar complextissue; and

FIG. 3 depicts an embodiment of an acellular human nipple areolarcomplex extracellular matrix scaffold (ECMS) originating from the samehuman native nipple areolar complex in FIG. 2.

FIG. 4 depicts a graph showing Relative Fluorescent Units (RFU)measurement of native versus processed tissue to show decreased immuneresponse when tissue is decellularized. MCP-1 (Monocyte HemotacticProtein-1) is used as positive control.

FIG. 5A depicts an illustration of a reconstructed breast mound aftermastectomy.

FIG. 5B depicts an illustration of a prepared area for a desired nipplelocation on the breast.

FIG. 5C depicts an illustration of an ECM scaffold of the presentinvention affixed in the desired location.

FIG. 5D depicts an illustration of a breast having an ECM scaffold ofthe present invention having cells repopulated by the patient to createan anatomical human nipple areolar complex.

FIG. 6A depicts a histological slice of native human nipple tissue at200× magnification stained with hematoxylin and eosin.

FIG. 6B depicts a histological slice of human nipple ECM scaffoldprepared using Example 1 methods at 200× magnification stained withhematoxylin and eosin.

FIG. 6C depicts a histological slice of human nipple ECM scaffoldprepared using Example 2 methods at 200× magnification stained withhematoxylin and eosin.

FIG. 6D depicts a histological slice of human nipple ECM scaffoldprepared using Example 3 methods at 200× magnification stained withhematoxylin and eosin.

FIG. 7A depicts a histological slice of native human nipple tissue at200× magnification stained with Trichrome Stain.

FIG. 7B depicts a histological slice of human nipple ECM scaffoldprepared using Example 1 methods at 200× magnification stained withTrichrome Stain.

FIG. 7C depicts a histological slice of human nipple ECM scaffoldprepared using Example 2 methods at 200× magnification stained withTrichrome Stain.

FIG. 7D depicts a histological slice of human nipple ECM scaffoldprepared using Example 2 methods at 200× magnification stained withTrichrome Stain.

FIGS. 8A-8H depict histological stains of native human nipple areolartissue (left) versus human nipple areolar ECMS (right) at 200×magnification. Trichrome stain was used in FIG. 8A, and FIG. 8E.Hematoxylin and eosin stain was used in FIG. 8B-8D and FIG. 8F-8H.

While the above-identified drawings set forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION

The following description provides exemplary embodiments only, and isnot intended to limit the scope, applicability, or configuration of thedisclosure. Rather, the following description of the exemplaryembodiments will provide those skilled in the art with an enablingdescription for implementing one or more exemplary embodiments. It willbe understood that various changes may be made in the function andarrangement of elements without departing from the spirit and scope ofthe disclosure as set forth in the appended claims.

Specific details are given in the following description to provide athorough understanding of the embodiments. However, it will beunderstood by one of ordinary skill in the art that the embodiments maybe practiced without these specific details. For example, systems,processes, and other elements in the instant disclosure may be shown ascomponents in block diagram form in order not to obscure the embodimentsin unnecessary detail. In other instances, well-known processes,structures, and techniques may be shown without unnecessary detail inorder to avoid obscuring the embodiments. Further, like referencenumbers and designations in the various drawings indicated likeelements.

Also, it is noted that individual embodiments may be described as aprocess which is depicted as a flowchart, a flow diagram, a data flowdiagram, a structure diagram, or a block diagram. Although a flowchartmay describe the operations as a sequential process, many of theoperations can be performed in parallel or concurrently. In addition,the order of the operations may be re-arranged. A process may beterminated when its operations are completed, but could have additionalsteps not discussed or included in a figure. Furthermore, not alloperations in any particularly described process may occur in allembodiments. A process may correspond to a method, a function, aprocedure, a subroutine, a subprogram, etc. When a process correspondsto a function, its termination corresponds to a return of the functionto the calling function or the main function.

The present invention presents a novel nipple areolar complex (NAC) andmethod for NAC replacement by utilizing a tissue-engineeredextracellular matrix scaffold created from human nipple areolar tissueto enable the patient to regenerate an anatomical nipple made from herown cells. By using an extracellular matrix derived from human nippleareolar tissue, the NAC of the present invention maintains the uniquestructural and growth factor environment of the nipple.

Previous in vitro data indicates that the removal of cellular componentsfrom native tissues leaves behind a complex protein matrix, designatedextracellular matrix, which can provide cells a combination of cues thatclosely resemble the in vivo environment in vitro. Recent studies havesuggested that superior function and complex tissue formation occurredwhen ECM scaffolds were derived from site-specific homologous tissuescompared with heterologous tissues.

In some embodiments of the present invention there are disclosed methodsfor processing or fabricating a human nipple areolar complexextracellular matrix scaffold (ECMS).

In another embodiment of the present invention, proteins remain behindin the scaffold (that aid in cellular in growth) following thedecellularization process. The ability for the human nipple areolarcomplex ECMS of the present invention to result in a patient'sreformation of a nipple areolar complex as opposed to current methodssuch as tattoos or nipple flap creation, such reformation includingadditional benefits such as enhanced stimuli response (like ability ofnipple to harden when exposed to temp changes or sensual stimuli) of thenipple areolar complex ECMS of the present invention versus traditionalnipple flap creation. While limited, increase of sensation of the nippleareolar complex is realized when utilizing the nipple areolar complexECMS of the present invention. In yet another embodiment of the presentinvention, the reformed nipple areolar complex utilizing the nippleareolar complex ECMS of the present invention results in increasedprojection as compared to traditional nipple areolar flap creation, aswell as increasing potential for pigmentation as compared to nippleareolar flap creation. The scaffolding network of the nipple areolarcomplex ECMS allows for formation of actual nipple versus scar/tattoo,including texture, projection and pigmentation.

Referring now to FIG. 1, the method comprises the steps of harvestinghuman nipple areolar complex tissue 10, treating the tissue with astabilizing medium 20, the stabilizing medium configured to prepare thetissue for transport to a processing center, decellularizing the tissue30 to form a nascent scaffold, and sterilizing the scaffold 40 to yielda human nipple areolar complex extracellular matrix scaffold (ECMS) fromthe tissue. As seen in FIG. 2 and FIG. 3, both the native human nippleareolar complex and the ECMS derived from said complex demonstratecharacteristics of native human nipple areolar complexes, specifically aprojecting nipple and areola.

Harvesting the tissue 10 may comprise harvesting human nipple tissuefrom cadavers under standard processes of tissue procurement. Harvestingmay be done in coordination with a tissue procurement company or atissue bank. A donor nipple may be harvested preferably under asepticconditions less than 48 hours after death with a dermatome, andmaintained at low temperature, preferably no warmer than 4° C. In anexemplary embodiment, human nipple tissue is subject to minimum tissuerequirements, including but not limited to: no previous indication ofbreast cancer or irradiation in breast as well as donor testing forinfectious agents, such as HIV, Hepatitis C, and Hepatitis B, asrecommended by the American Association of Tissue Banks.

Following harvesting 10, the tissue is treated and stabilized 20 fortransport with a stabilizing solution. The stabilizing solution may (1)prevent osmotic, hypoxic, autolytic and proteolytic degradation to thetissue, (2) protect against microbial contamination of the tissue, and(3) reduce mechanical damage to the tissue. The stabilizing solution maycomprise one or more of a buffer, antioxidants, oncotic agents, proteaseinhibitors, antibiotics, and optionally a smooth muscle relaxant. Thetissue may stay in the stabilizing solution for no more than 7 days, butideally the tissue is transported by overnight delivery on wet ice to atissue processing center.

After the tissue is stabilized 20 and transported to a processingfacility, the tissue is decellularized 30. Historically,decellularization may be accomplished using a number of chemicaltreatments, including incubation in certain salts, detergents orenzymes. The use of the detergent TRITON X-100, a trademarked product ofRohm and Haas Company of Philadelphia, Pa., has been demonstrated toremove cellular membranes, as detailed in U.S. Pat. No. 4,801,299. Otheracceptable decellularizing detergents include polyoxyethylene (20)sorbitan mono-oleate and polyoxyethylene (80) sorbitan mono-oleate(Tween 20 and 80), sodium deoxycholate,3-[(3-chloramidopropyl)-dimethylammino]-1-propane-sulfonate,octyl-glucoside and sodium dodecyl sulfate.

The foregoing decellularization process is exemplary and is non-limitingwith regard to the decellularization phase of the present invention.Alternatively, enzymes may be used to accomplish decellularization,including but not limited to dispase II, trypsin, and thermolysin.Trypsin and other non xeno-free enzymes may be used in preclinicalstudies as a comparison tool to compare decellularization efficiency.These enzymes react with different components of collagen andintercellular connections in achieving their effects. Dispase II attacksType IV collagen, which is a component of the lamina densa and anchoringfibrils of the basement membrane. Thermolysin attacks the bulbousphemphigoid antigen in the hemidesmosome of the basal layer ofkeratinocytes. Trypsin attacks the desmosome complex between cells. Dueto the proteolytic nature of these enzymes, care must be taken thatcellular removal occurs without significant damage to the extracellularmatrix, including the basement membrane complex. This is a function ofconcentration, time and temperature. If used for too long a time or attoo high a concentration, dispase II for example can completely removethe basement membrane complex from the dermis.

For example, with human cadaver skin Dispase II at 1.0 units/ml for 90minutes at 37° C. will remove all keratinocytes except the basal layer,while some damage is already occurring to the basement membrane complex.Thermolysin at 200 ug/ml for 30 minutes at 4° C. will essentially removeall keratinocytes without damage to the basement membrane complex onsome occasions, but this varies from donor to donor with evidence ofbasement membrane damage being seen in some donors. Incubation of skinin 1 molar sodium chloride for 16 hours for human skin and 48 hours forporcine skin will routinely allow clean separation of the epidermis anddermis without damage to the basement membrane complex.

In addition to salts, detergents and enzymes, the processing solutionalso contains certain protease inhibitors, to prevent degradation of theextracellular matrix. Collagen-based connective tissues containproteases and collagenases as endogenous enzymes in the extracellularprotein matrix. Additionally, certain cell types including smooth musclecells, fibroblasts and endothelial cells contain a number of theseenzymes inside vesicles called lysosomes. When these cells are damagedby events such as hypoxia, the lysosomes are ruptured and their contentsreleased. As a result, the extracellular matrix can undergo severedamage from protein, proteoglycan and collagen breakdown. This damagemay be severe, as evidenced in clinical cases of cardiac ischemia wherea reduction in oxygen which is insufficient to cause cell death resultsin pronounced damage to the collagen matrix. Additionally, a consequenceof extracellular breakdown is the release of chemoattractants, whichsolicit inflammatory cells, including polymorphonuclear leukocytes andmacrophages, to the graft, which are intended to remove dead or damagedtissue. These cells also, however, perpetuate the extracellular matrixdestruction through a nonspecific inflammatory response. Accordingly,the processing solution contains one or more protease inhibitorsselected from the group of N-ethylmaleimide (NEM),phenylmethylsulfonylfluoride (PMSF) ethylenediamine tetraacetic acid(EDTA), ethylene glycol-bis-(2-aminoethyl(ether)NNN′N′-tetraacetic acid,ammonium chloride, elevated pH, apoprotinin and leupeptin to preventsuch damage.

In addition to salts, detergents, enzymes and protease inhibitors, theprocessing solution generally contains an appropriate buffer. This mayinvolve one of many different organic buffers which are described above.The inventors prefer to use an organic buffer selected from the groupconsisting of 2-(N-morpholino)ethanesulfonic acid (MES), Tris(hydroxymethyl)aminomethane (TRIS) and(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES).Alternatively, a low salt or physiological buffer including phosphatebicarbonate acetate citrate glutamate with or without glycine, may bemore appropriate in certain applications. Low salt or physiologicalbuffers are more able to support the infiltration of the graft withviable cells and hence are more relevant when cellular infiltrationincluding neovascularization is essential to early survival of the graftas in transplanted dermal matrix.

As the processing solution may contain chemicals that would beirritating or inflammatory on transplantation, it is important that theprocessing solution be thoroughly rinsed from the tissue. In someembodiments, this washing occurs by rinsing in sufficient changes ofappropriate buffer, until residues of the processing solution arereduced to levels compatible with transplantation. Alternatively,components of the processing solution may be neutralized by specificinhibitors, e.g., dispase II by ethylenediaminetetraacetic acid (EDTA)or trypsin by serum.

In some embodiments, decellularizing human nipple tissue 30 yields adecellularized extracellular matrix (ECM) of an existing human nippletissue, the decellularized matrix aiding in the long-term maintenance ofa reconstructed human nipple architectural structure. The ECM is acompilation of secretions from various cell types that make up eachrespective tissue and has been shown to aid in the cell proliferation,migration, and differentiation. Previous in vitro data indicates thatthe removal of cellular components from native tissues leaves behind acomplex protein matrix, designated extracellular matrix, which canprovide cells a combination of cues that closely resemble the in vivoenvironment in vitro. Recent studies have demonstrated that superiorfunction and complex tissue formation occurs when ECM scaffolds werederived from site-specific homologous tissues compared with heterologoustissues. Therefore, preserving the composition and architecture of thehuman nipple areolar ECM during the process of decellularization 30 isof utmost importance.

Typically, when an allograft tissue is implanted, a strong immunologicalresponse occurs within the body. This immune reaction (mainly driven bymonocytes) can cause rejection, necrosis and/or extrusion of theimplanted material. However, when the allografts are stripped of nativecell proteins and nucleic acids (not ECM proteins), the immune responseis attenuated. Hence, the removal of native non-ECM cell proteins andnucleic acids from the allograft is used as a strategy to guarantee thesuccessful integration of allografts, without eliciting strong immunereactions. An example of the reduced immune response through the processof decellularization as compared to native tissue can be seen in FIG. 4.FIG. 4 presents a graph showing Relative Fluorescent Units (RFU)measurement of native versus processed tissue, showing decreased immuneresponse when tissue is decellularized. MCP-1 (Monocyte HemotacticProtein-1) is used as positive control. MTF is a brand of commerciallyavailable acellular skin. Native is native porcine tissue. Processed istissue processed to decellularize the tissue using similar methodsclaimed herein. Processed tissue shows decrease in monocytechemoattraction (or immune response) as compared to all other materials.

In one embodiment, the human nipple areolar complex ECMS of the presentinvention exhibits reduced immunogenicity by utilization of a fullydecellularized graft versus native tissue.

In order to reasonably ascertain whether decellularized tissue willelicit a strong negative immune reaction in human patients, a monocytechemotactic assay was performed. Chemotaxis (migration) of humanmonocytes/macrophages, which are the first responders to tissue injury,were measured in decellularized versus native tissue adipose tissue toevaluate the response of these cells in vitro (see FIG. 4). When lookingat decellularized tissue, chemo-attraction values of human monocytes(first responders to tissue injury) were significantly lower than thosemeasured in native tissue, showing the decellularization process reducesinnate immunogenicity.

Referring again to FIG. 1, decellularizing the tissue 30 comprisesprocessing and preserving human nipple areolar tissue fortransplantation, wherein the processing and preserving steps maycomprise chemical pretreatment, cell removal, and sterilization,yielding a human nipple areolar scaffold. The processing step isdesigned to generate a transplantable biological tissue graft, whereinthe graft (a) provides an extracellular matrix which can be regeneratedand remodeled by the host, (b) does not elicit an negative immuneresponse by the host, and (c) can be easily stored and transported. Thepreserving step may comprise incubating the tissue in a decellularizingsolution configured to remove antigenic donor cells within the tissue.Decellularizing the tissue 30 may further comprise optimizing theconcentration and time duration for cellular removal while preservingextracellular matrix integrity of the ECM. After the cells arethoroughly removed from the tissue, the tissue is washed several timesto ensure removal of any remaining detergent or decellularizingsolution. Decellularization of the tissue 30 may be verified byvisualizing nuclear material in tissue sections stained with4′,6-diamidino-2-phenylindole (DAPI) or hematoxylin and eosin (H&E).Additionally, remaining double stranded DNA can be quantified.Decellularizing solutions may comprise buffers, antibiotics, detergents,protease inhibitors and/or enzymes.

Once the nipple areolar complex ECMS has been decellularized 30, anipple areolar complex ECMS may then be sterilized 40. In oneembodiment, the nipple areolar complex ECMS of the present invention isnot sterilized, or lacks a sterilization step. In some embodiments whichinclude a sterilization step, the scaffold is sterilized 40 with acombination of peracetic acid and supercritical carbon dioxide or acombination of peracetic acid and ethanol. In some embodiments, thescaffold may be sterilized using gamma irradiation, ethylene oxide,and/or electron beam irradiation. The sterilizing step 40 yields an ECMSthat is ready for implantation.

In Vivo Recellularization. Turning to FIG. 5A-E, various stages of theregeneration process for the nipple areolar complex comprising the humannipple areolar complex ECMS of the present invention are presented. FIG.5A provides an illustration of a reconstructed breast mound aftermastectomy. FIG. 5B provides a prepared portion of a female breast in alocation desired for a nipple areolar complex ECMS of the presentinvention to be administered. In a preferred embodiment, this occurs byde-epitheliating the skin in the location where the nipple will belocated. FIG. 5C presents the administration of a human nipple areolarcomplex ECMS of the present invention being sutured or adhesivelyapplied to the patient directly. The nipple areolar scaffold may beapplied to the recipient directly, whether autologous or allogeneic. Thenipple areolar scaffold is then secured in place for incorporation intothe donor tissue. May be used alone or in combination with othertreatments both cellular and non-cellular deemed appropriate by thephysician.

Blood, plasma proteins, and cells are absorbed into the scaffoldallowing cellular access to the scaffold. Neutrophils infiltrate thescaffold and initiate the process of tissue remodeling. Tissueremodeling continues with infiltration of macrophages into the scaffoldfollowed by stem cell recruitment and proliferation within the scaffold.As shown in FIG. 5D, cells from the patient repopulate the nippleareolar complex ECMS of the present invention to create an anatomicallycorrect nipple areolar complex. During the final stages ofrecellularization, stem cells within the scaffold may differentiate intothe appropriate cell types, directed by signaling molecules within thescaffold. FIG. 5E presents the nipple areolar complex ECMS of thepresent invention wherein the growth factor environment within the ECMSpromotes natural pigmentation and improved projection. In an alternativeembodiment, additional materials may be added to aid inrevascularization of the scaffold such as microvascular fragmentsisolated from adipose tissue of the recipient.

In another embodiment, the nipple areolar complex ECMS of the presentinvention is capable of being implanted in a single procedure, andwherein the implantation will lead to cellular incorporation through thepatient's innate cellular response.

In Vitro Recellularization. The nipple areolar scaffold may bere-cellularized before implantation using xeno-free culturing methodswith cells from the recipient. The cells may include but are not limitedto fibroblasts, epithelial cells, mammary endothelial cells, mammaryepithelial cells, vascular smooth muscle cells, bone marrow mesenchymalstems cells, adipose derived stem cells, or induced pluripotent stemcells. If using epithelial cells, the cells are isolated from thepatient through a skin scraping. The cells will be cultured and expandedin the laboratory using an appropriate medium such as but not limited tokeratinocyte serum-free media or other relevant xeno-free medias. Oncesufficient cell expansion has occurred, the cells may be lifted forcollection by adding a xeno-free cell detachment solution, such as butnot limited to ACCUTASE (Stemcell Technologies), TrypLE Select(ThermoFisher Scientific) or ACCUMAX (EMD Millipore), to the media untilsufficient cell lifting has occurred. The cells are removed from thetissue culture plate and placed in a conical tube for centrifugation.Total cell count is calculated and appropriate volume of cells and mediais added to the scaffold to obtain sufficient cell density per area ofscaffold for optimal cell repopulation.

EXAMPLES

The below examples set forth non-limiting embodiments, and for eachexample, stringency will be modified on how tolerant the tissue is ofthe various decellularization procedures. These procedures can be usedfor either autologous or allogeneic tissues.

Example 1

A donor nipple areolar complex (as seen in FIG. 2) is harvested underaseptic conditions with a dermatome, and maintained at low temperature,preferably no warmer than 4° C. The tissue is treated and stabilized fortransport with a stabilizing solution. This solution may contain cellculture medium containing 1% penicillin, 1% streptomycin, 1%amphotericin b, 1% antibiotic/antimycotic, 5% heparin, and 2% melatonin.The tissue may stay in the stabilizing solution for no more than 7 days.

The tissue is removed from stabilizing solution and the fat and muscletissue that remains beneath the human nipple areolar tissue is removed.This may be done immediately or just before processing. The tissue isthen placed in a vacuum bag to prevent freezer burn and the excess airis removed.

The tissue is then placed in −30° Celsius and freeze-thawed up to 3times. The tissue is then placed in a conical tube or Erlenmeyer flaskand exposed to 1% sodium dodecyl sulfate (SDS) diluted in PBS or HanksBalanced Salt Solution while agitated at 175 RPM. The solutions arechanged every 12-24 hours. The solutions may also contain 1%antibiotic/antimycotic if deemed necessary. The tissue will be exposedto the decellularization solution for 3-14 days depending on the age ofthe donor tissue was collected from. The tissue will be agitated insolution at 37±3° C. at 175 RPM. The solution may be changed every 12-24hours or when solution saturation is reached, whichever comes first.Tissue under the age of 45 years may need to be exposed todecellularization solutions longer due to thicker collagen base ofyounger human tissue.

After exposure to decellularizing solutions, the tissue is thoroughlyrinsed by agitating in deionized or Millipore water at 175 RPM for 3hours. This process may be repeated 3 times. Decellularized tissue maybe stored in 4° C. or −30° C. either in phosphate buffered saline (PBS)or a vacuum bag until such time as sterilization occurs.

Sterilization is performed by exposing the human nipple areolar ECMS toa mixture of 0.1% peracetic acid and 4% ethanol for up to two hours.

Example 2

A donor nipple areolar complex (as seen in FIG. 2) is harvested underaseptic conditions with a dermatome, and maintained at low temperature,preferably no warmer than 4° C. The tissue is treated and stabilized fortransport with a stabilizing solution. This solution may contain cellculture medium containing 1% penicillin, 1% streptomycin, 1%amphotericin b, 1% antibiotic/antimycotic, 5% heparin, and 2% melatonin.The tissue may stay in the stabilizing solution for no more than 7 days.

The tissue is removed from stabilizing solution and the fat and muscletissue that remains beneath the human nipple areolar tissue is removed.This may be done immediately or just before processing. The tissue isthen placed in a vacuum bag to prevent freezer burn and the excess airis removed.

The tissue is then placed in −30° Celsius and freeze-thawed up to 3times. The tissue is then placed in a conical tube or Erlenmeyer flaskand exposed to 1% sodium dodecyl sulfate (SDS), 5% TRITON-X 100, and 1%Ammonium Hydroxide diluted in PBS or Hanks Balanced Salt Solution whileagitated at 175 RPM. The solutions are changed every 12-24 hours. Thesolutions may also contain 1% antibiotic/antimycotic if deemednecessary. The tissue will be exposed to the decellularization solutionfor 3-14 days depending on the age of the donor tissue was collectedfrom. The tissue will be agitated in solution at 37±3° C. at 175 RPM.The solution may be changed every 12-24 hours or when solutionsaturation is reached, whichever comes first. Tissue under the age of 45years may need to be exposed to decellularization solutions longer dueto thicker collagen base of younger human tissue.

After exposure to decellularizing solutions, the tissue is thoroughlyrinsed by agitating in deionized or Millipore water at 175 RPM for 3hours. This process may be repeated 3 times. Decellularized tissue maybe stored in 4° C. or −30° C. either in phosphate buffered saline (PBS)or a vacuum bag until such time as sterilization occurs.

Sterilization is performed by exposing the human nipple areolar ECMS toa mixture of 0.1% peracetic acid and 4% ethanol for up to two hours.

Example 3

A donor nipple areolar complex (as seen in FIG. 2) is harvested underaseptic conditions with a dermatome, and maintained at low temperature,preferably no warmer than 4° C. The tissue is treated and stabilized fortransport with a stabilizing solution. This solution may contain cellculture medium containing 1% penicillin, 1% streptomycin, 1%amphotericin b, 1% antibiotic/antimycotic, 5% heparin, and 2% melatonin.The tissue may stay in the stabilizing solution for no more than 7 days.

The tissue is removed from stabilizing solution and the fat and muscletissue that remains beneath the human nipple areolar tissue is removed.This may be done immediately or just before processing. The tissue isthen placed in a vacuum bag to prevent freezer burn and the excess airis removed.

The tissue is then placed in −30° Celsius and freeze-thawed up to 3times. The tissue is then placed in a conical tube or Erlenmeyer flaskand exposed to 5% TRITON-X 100 and 1% Ammonium Hydroxide diluted in PBSor Hanks Balanced Salt Solution while agitated at 175 RPM. The solutionsare changed every 12-24 hours. The solutions may also contain 1%antibiotic/antimycotic if deemed necessary. The tissue will be exposedto the decellularization solution for 3-14 days depending on the age ofthe donor tissue was collected from. The tissue will be agitated insolution at 37±3° C. at 175 RPM. The solution may be changed every 12-24hours or when solution saturation is reached, whichever comes first.Tissue under the age of 45 years may need to be exposed todecellularization solutions longer due to thicker collagen base ofyounger human tissue.

After exposure to decellularizing solutions, the tissue is thoroughlyrinsed by agitating in deionized or Millipore water at 175 RPM for 3hours. This process may be repeated 3 times. Decellularized tissue maybe stored in 4° C. or −30° C. either in phosphate buffered saline (PBS)or a vacuum bag until such time as sterilization occurs.

Sterilization is performed by exposing the human nipple areolar ECMS toa mixture of 0.1% peracetic acid and 4% ethanol for up to two hours.

Example 4

In an alternative embodiment, sterilization is performed using acombination of peracetic acid and supercritical carbon dioxide (SCCO₂)at concentrations and durations optimized for NAC tissue. Thesterilization of the NAC extracellular matrix tissue is carried out in a20-liter pressure chamber which houses wire baskets for holding packagedsamples as well as an additive pad to which poly acrylic acid (PAA)(Sigma, ˜24-50% vol./vol. PAA in acetic acid) can be added if necessary.The pressure and temperature of the SCCO₂ is held constant at 7000-10000kPa and 30-37° C., respectively, whereas the amount of PAA and theduration of exposure to SCCO₂ will be varied. An additive of peraceticacid may be used to ensure sterility if found appropriate from ˜10-40%vol/vol in concentration (Sigma Aldrich). The pressure may be held at7000-10000 kPA at temperatures ranging from 30-37° C. The amount ofperacetic acid may vary as well as the duration of tissue exposure (10to 140 minutes).

Whereas many alterations and modifications of the present invention willno doubt become apparent to a person of ordinary skill in the art afterhaving read the foregoing description, it is to be understood that theparticular embodiments shown and described by way of illustration are inno way intended to be considered limiting. Further, the disclosure,examples, and related figures have been described with reference toparticular preferred embodiments, but variations within the spirit andscope of the disclosure will occur to those skilled in the art.

In support of the foregoing Examples FIGS. 6-8 present various aspectsof the present invention. FIG. 6A presents a histological slice ofnative human nipple tissue at 200× magnification stained withhematoxylin and eosin. Nuclei are stained evident through purplestaining, while the ECM is stained pink. In native human nipple tissue,the cornified keratinocyte layer is visible at the surface withstratified epithelial cells within the epidermis. Melanocytes areprimarily localized to the basal layer of the epidermis, although,unique to nipple areolar tissue, some melanocytes are also presentwithin the dermal layers.

FIG. 6B presents a histological slice of human nipple ECM scaffoldprepared using Example 1 methods at 200× magnification stained withhematoxylin and eosin. As compared to native human nipple tissue, thenuclei and epithelial cell layers have been removed and only the ECMremains.

FIG. 6C presents a histological slice of human nipple ECM scaffoldprepared using Example 2 methods at 200× magnification stained withhematoxylin and eosin. As compared to native human nipple tissue, thenuclei and epithelial cell layers have been removed and only the ECMremains.

FIG. 6D presents a histological slice of human nipple ECM scaffoldprepared using Example 3 methods at 200× magnification stained withhematoxylin and eosin. As compared to native human nipple tissue, thenuclei and epithelial cell layers have been removed and only the ECMremains.

FIG. 7A presents a histological slice of native human nipple tissue at200× magnification stained with Trichrome Stain. Nuclei are stainedblack; cytoplasm, muscle, and erythrocytes stained red; and collagenstained blue. The epidermal layer of the tissue is intact, with theepithelial layer removed. FIG. 7B presents a histological slice of humannipple ECM scaffold prepared using Example 1 methods at 200×magnification stained with Trichrome Stain. FIG. 7C presents ahistological slice of human nipple ECM scaffold prepared using Example 2methods at 200× magnification stained with Trichrome Stain. FIG. 7Dpresents a histological slice of human nipple ECM scaffold preparedusing Example 3 methods at 200× magnification stained with TrichromeStain.

FIG. 8 presents histological stains of native human nipple areolartissue (left) versus human nipple areolar ECMS (right) at 200×magnification. Trichrome stain was used in FIG. 8A, and FIG. 8E.Hematoxylin and eosin stain was used in FIG. 8B-D and FIG. F-H.Corresponding structures are evident in both the native anddecellularized tissue. H&E staining shows unique microstructure of thehuman nipple is maintained despite cell removal.

It is noted that the foregoing examples have been provided merely forthe purpose of explanation and are in no way to be construed as limitingof the present invention. While the present invention has been describedwith reference to exemplary embodiments, it is understood that thewords, which have been used herein, are words of description andillustration, rather than words of limitation. Changes may be made,within the purview of the appended claims, as presently stated and asamended, without departing from the scope and spirit of the presentinvention in its aspects. Although the present invention has beendescribed herein with reference to particular means, materials andembodiments, the present invention is not intended to be limited to theparticulars disclosed herein; rather, the present invention extends toall functionally equivalent structures, methods and uses, such as arewithin the scope of the appended claims.

1. A method for regenerating a nipple areolar complex comprising: a. harvesting human nipple areolar complex tissue; b. treating the tissue with a stabilizing medium, wherein the stabilizing medium stabilizes the tissue during a transport; c. decellularizing the tissue, wherein the cells and DNA are removed; and d. sterilizing the tissue; wherein said method yields a nipple areolar complex extracellular matrix scaffold (ECMS) comprised of at least dermal or subdermal layers and wherein the human nipple areolar complex ECMS has reduced immunogenicity in humans as compared to native tissue.
 2. (canceled)
 3. The method of claim 1, wherein the harvesting step comprises harvesting the tissue from a cadaver.
 4. The method of claim 1, wherein the harvesting step comprises harvesting the tissue from an individual during a surgery.
 5. The method of claim 1, wherein the decellularizing step comprises decellularization using one or more decellularization methods selected from a group consisting of detergents, enzymes, salts, electrophoresis, and combinations thereof.
 6. The method of claim 1 further comprising repopulating the scaffold by cells in vivo after application of the nipple areolar complex ECMS to an individual.
 7. The method of claim 6, wherein the step of repopulating the scaffold comprises repopulating the nipple areolar complex ECMS with exogenous cells selected from a group consisting of fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stem cells, adipose derived stem cells, induced pluripotent stem cells, and combinations thereof.
 8. A method of treatment comprising a. identifying an individual with a need for a nipple reconstruction; b. forming a nipple areolar complex extracellular matrix scaffold (ECMS), the forming comprising: i. harvesting a human nipple areolar complex tissue; ii. treating the tissue with a stabilizing medium, wherein the medium stabilizes the tissue during transport; iii. decellularizing the tissue, wherein cells and DNA are removed; and c. sterilizing the tissue; wherein said method yields a sterile nipple areolar complex ECMS comprising at least dermal or subdermal layers; and d. applying the nipple areolar complex ECMS to the individual.
 9. The method of claim 8, wherein the individual is an individual who has lost a nipple due to a cancer, a trauma, congenital absence, or a cosmetic reason.
 10. The method of claim 8, wherein the decelluarizing step comprises decellularization using one or more decellularization methods selected from a group consisting of detergents, enzymes, salts, electrophoresis, and combinations thereof.
 11. The method of claim 8, wherein the applying step comprises direct implantation of the ECMS onto the individual.
 12. The method of claim 8, wherein the implanted decellularized ECMS is repopulated in vivo by the patient's native cells.
 13. The method of claim 8 further comprising, before the applying step, recellularizing the ECMS to yield a recellularized ECMS, wherein the applying step comprises applying the recellularized ECMS to the individual.
 14. The method of claim 13, wherein the recellularization of the applying step comprises recellularizing the nipple areolar complex ECMS with exogenous cells selected from a group consisting of fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stem cells, adipose derived stem cells, induced pluripotent stem cells, and combinations thereof.
 15. A nipple areolar complex extracellular matrix scaffold (ECMS), comprising a decellularized human nipple areolar complex ECMS, wherein the nipple areolar complex ECMS is capable of supporting regeneration of a nipple areolar complex when implanted on a patient without generating an negative immune response in said patient
 16. A nipple areolar complex ECMS of claim 15, wherein the nipple areolar complex ECMS is capable of supporting growth of melanocytes for pigmentation of the nipple areolar complex ECMS.
 17. A nipple areolar complex ECMS of claim 15, wherein the nipple areolar complex ECMS comprises at least dermal or subdermal layers.
 18. A nipple areolar complex ECMS of claim 15, wherein said nipple areolar complex ECMS is further capable of increased patient sensation of the nipple areolar complex occurring within 2 years of ECMS implantation on said patient as compared to surgical flap nipple reconstruction.
 19. A nipple areolar complex ECMS of claim 15, wherein said nipple areolar complex ECMS is further capable of maintaining projection of the nipple after implantation on a patient.
 20. A nipple areolar complex ECMS of claim 15, wherein said nipple areolar complex ECMS is capable of generating pigmentation after implantation on a patient.
 21. The method of claim 1, wherein the human nipple areolar complex ECMS comprises a basement membrane complex.
 22. The method of claim 8, wherein the human nipple areolar complex ECMS comprises a basement membrane complex. 